Urokinase-type plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. In addition to its proteolytic properties, uPA induces cellular migration and activation of intracellular signaling pathways through mechanisms that are independent of proteolysis. uPA potentiates neutrophil functions important for host defense, including priming for superoxide production and chemotaxis. uPA enhances LPS-induced neutrophil responses, including proinflammatory cytokine expression, activation of intracellular signaling pathways involving the kinases Akt and c-Jun N-terminal kinase (JNK), and nuclear translocation of NF-κB. Transgenic mice unable to produce uPA have been shown to be protected from endotoxemia-induced, neutrophil dependent lung injury.
uPA is secreted by many cell populations, including neutrophils, endothelial and epithelial cells, as a single-chain proenzyme that possesses little or no proteolytic activity. Single chain uPA (scuPA) is converted after single cleavage between Lys158 and Ile159 into the proteolytically active dual chain enzyme, consisting of an NH2-terminal A chain and a proteolytic domain-containing B chain. uPA is composed of three structurally independent components: (1) a growth factor domain (GFD, amino acids 1-46), (2) a kringle domain (KD, amino acids 47-135), and (3) a proteolytic domain (PD, amino acids 159-411). The GFD is responsible for the interaction of urokinase with the uPAR/CD87 receptor. The PD includes the catalytically active site of the enzyme. Enzymatic digestion of single chain uPA (scuPA) yields an amino terminal fragment (ATF), which consists of the GFD and KD, and a low molecular weight fragment (LMW-uPA). Both single chain and cleaved, two chain uPA, as well as the amino terminal fragment (ATF) of uPA, which is contains the GFD, can bind to the uPAR/CD87 receptor. uPA lacking the GFD fails to interact with uPAR/CD87, but can bind to other cell surface receptors, including integrins and those belonging to the low density lipoprotein receptor (LDLR) family either through the KD or PD.
Human uPA also enhances inflammatory responses mediated by activation of murine neutrophils exposed to sub-maximal stimulatory doses of LPS. The fact that human uPA is unable to bind to murine uPAR suggests that receptors other than uPAR are involved in potentiating neutrophil responses. Moreover, because the uPA GFD is known to be responsible for interactions with uPAR, uPA domains other than GFD are likely responsible for the proinflammatory effects of uPA on neutrophils.
Understanding the mechanism by which uPA mediates its proinflammatory activities could provide important information useful in selectively inhibiting uPA mediated inflammation, while retaining thrombolytic activities of uPA mediated by other mechanisms.
There is an undeveloped need to identify new agents that specifically bind or uPA and inhibit uPA mediated inflammation. Such agents would be useful for diagnostic screening and therapeutic intervention in disease states that are associated with uPA activity.
Accordingly, it is an object of the present invention to provide specific binding agents of uPA that inhibit uPA mediated inflammation. Such agents of the present invention take the form of antibodies and fragments thereof that specifically bind to uPA epitopes.
The disclosure all patents, patent applications, and other documents cited herein are hereby expressly incorporated by reference in their entirety.